Autosomal dominant polycystic kidney disease (ADPKD) causes the formation of cysts that expand progressively and disrupt the structure of the renal parenchyma leading to a dramatic loss in kidney function. Two genes are responsible for the ADPKD phenotype (PKD1 and PKD2) although the majority of patients have mutations in PKD1, which encodes the polycystin 1 protein (PC1). PC1 is an integral membrane protein shown to localize to the primary cilium in renal epithelial cells. There is evidence that it acts a sensor for fluid flow and pressure in the kidney. PC1 undergoes proteolytic cleavage of its cytoplasmic C-terminal tail (PC1-CTT), which is then released and can translocate to the nucleus to regulate gene expression. The goal of this application is to identify the mechanism and functional activity of PC1-CTT cleavage in order to better understand the role of PC1 in the pathogenesis of ADPKD. This will expose new molecular targets for therapeutic intervention to provide treatment for ADPKD patients. Data from our lab indicates that overexpression of exogenous PC1-CTT can rescue phenotypes caused by loss of full length PC1 in both in vitro and in vivo model systems. Through regulation of gene expression the PC1-CTT can influence a number of signaling pathways that control cell proliferation and apoptosis. In Aim 1, we will identify the PC1-CTT cleavage site using Edman degradation and generate a mutant construct of PC1 where C-terminal cleavage is disrupted. The cleavage incompetent status of this mutant will be assessed by Western blot. We will test the functional consequences of the loss of PC1-CTT cleavage by determining whether, in contrast to wild type PC1, the mutant PC1 fails to rescue the cystic phenotype of PKD-/- cells grown in a 3D cell culture system. In Aim 2, we will study the interaction between PC1-CTT and the transcriptional co-activator TAZ through in vitro biochemical studies as well as genetic interaction experiments in zebrafish. We have identified that the PC1-CTT can bind to and stimulate the activity of TAZ. This interaction is likely relevant to ADPKD pathogenesis since TAZ knockout mice develop polycystic kidney disease. The minimal domain of TAZ required to immunoprecipitate the PC1-CTT will be defined through the generation of chimeric proteins of TAZ and its close structural homolog YAP, which cannot robustly bind the PC1-CTT. We will also identify whether the PC1-CTT stimulates the activity of TAZ by altering its subcellular localization or by increasing its affinity for target proteins and genes within the nucleus. For in vivo studies, zebrafish treated with morpholinos that knockdown the expression of PC1 will be used as a model of ADPKD. These fish develop a severe dorsal tail curvature phenotype that can be rescued with the expression of PC1- CTT mRNA. We hypothesize that TAZ morpholino knockdown will prevent this rescue by the PC1-CTT and that expression of a constitutively active mutant of TAZ may be sufficient to restore the wild type phenotype in the absence of the PC1-CTT.